Archive for the ‘Microscopes’ Category

Size of Field of a Microscope.
Size of Field of a Microscope.

Size of Field Microscope

Quite a general but errorneous idea prevails that the size of the tube has an influence on the size of the field. Except in eye-pieces of very low power, or with tubes with smaller than usual dimensions, this is not so. It must be remembered that a Huyghenian eye-piece admits of a definite size of field, and this is regulated by the opening in the diaphragm ; the same size of opening is used in all of the same power, whether it is an eye-piece for a large or small diameter.

A misconception also exists as to the definition of field. Such inquiries are often made as : " As we understand it, a wide-angle objective gives a larger field ? " but it does nothing of the kind. The angular aperture has no bearing whatever on the size of the field, The field of view, or that which is shown of the object's surface, is determined by the power of the objective and eye-piece.

Information on Microscope Optics.

Information on Microscope Optics.

Microscope Optics

The microscope is an optical instrument designed for the purpose of enlarging details to such an extent that they may be clearly discerned by the eye. It may be simple or compound, depending upon whether it contains one or more lenses. A simple microscope is usually found in the form of one double convex lens and is commonly called a magnifying glass. The compound microscope differs from this in that it has several lenses, each magnifying the image of the other until great enlargement is secured. It consists essentially of one lens, the objective, close to the subject, which forms an image which is in turn magnified by another lens, the ocular or eyepiece. Reference to Fig. I will help to make this clear.

In this diagram the objective O forms an image of the specimen on the slide S, at the plane PI. This image, if allowed to reach the eye, would be inverted and a real image. Before it can be formed, however, the light rays encounter the lower lens of the eyepiece B which, in com- bination with the upper lens C, produces a magnified virtual image at the plane P 2 , corresponding to the real image P^ Thus the magnifying power is the product of the separate magnifying powers of the two lens systems, or that of the objective multiplied by that of the eyepiece.

Thus it would appear that any magnification desired, however great, could be secured simply by increasing the magnifying power of the two lens systems. It would seem at first sight that there is no limit to the amount of detail we could perceive, for could we not use another micro- scope to magnify the image produced by the first and thus secure unlimited magnification? Magnification yes, but detail no, for, unfortunately, the amount of detail which may be discerned is limited by optical laws. Mere mag- nification of the subject does not enable us to see more detail. This quality, known as resolving power, is de- termined by the construction of the objective.

Fig. I. Diagram of the light path through a microscope.

An eyepiece with a magnification of ten times, that is, one which magnifies the objective image ten times, will give about all the detail that the objective is capable of resolving. This limit of resolving power is fixed by the nature of light itself. Light is not a continuous flow of substance. It consists of definite waves of definite wave- length. This gives to light, in a manner of speaking, a certain structure which makes it impossible to see things that are smaller than the structure of light itself. Re- solving power may be defined as the distance by which two small elements in an object must be separated in order to be visible, and is a function of what is known tfs the numerical aperture of the lens.

In microscopical writings the term numerical aperture is abbreviated to N.A. 'The higher the N.A. the greater the resolving power and the finer the detail which is re- vealed. Numerical aperture is equal to the effective aperture of the back lens of the objective divided by twice the equivalent focus. Thus if a very narrow pencil of light is used for illumination, the finest detail which may be revealed by a microscope of sufficient magnification is equal to ^L in which wl is the wavelength of the light used for illumination. As the pencil of light becomes wider, the resolving power is increased until a maximum is reached when the whole aperture is filled with light. In this case the resolving power is twice as great, as repre- sented by the formula 2 -^ ] A – This same limit is reached when a narrow pencil of light enters the lens as obliquely as possible. The wavelength of light may be taken as one half of i / 1,000 of a millimeter, or about i 750,000 of an inch. If then we assume a lens in which the effective aperture of the back lens is equal to the equivalent focus, the lens will have an N.A. of 0.5. This lens can separate lines which are 1/25,000 of an inch apart if the back lens is filled with light, but if a narrow pencil is used the lines must be only 1/12,500 of an inch apart to be resolved by this objective. So we see that extremely high resolving power requires objectives of wide numerical aperture, in the order of i.o N.A., which will resolve 50,000 lines to the inch. Use of such objectives calls for special equip- ment and manipulation.

In using a microscope we look through a sheet of glass, the cover glass over the specimen. While this is trans- parent it may act also as a reflector if the light passing through it strikes it at an angle greater than a certain fixed angle. For the same reason, and more readily be- cause of the black background provided by the inside of the microscope tube, the lens of the objective may become a reflector. In order to control this angle and provide a definite path for the light to travel we equip the micro- scope with a condenser (the substage condenser) placed in the path of the light. Thus we may control the light and regulate the amount so that it just fills the rear ele- ment of the objective when it is examined with the eye- piece removed from the tube. We also place a drop of oil on the cover glass and immerse the objective in this. The oil, having the same refractive index as the glass, presents a homogeneous material through which the light may travel in its own medium, thereby preventing the dis- persion which would otherwise take place. Such lenses are known as oil-immersion lenses and by their use nu- merical apertures as high as 1.4 may be attained, which, under the most favorable conditions, permit resolutions of the order of 100,000 lines to the inch. These objectives are available only on the most expensive professional microscopes, so the beginner need not search for them as accessory equipment to amateur instruments. This di- gression into numerical aperture is included solely for the fortunate possessors of more pretentious micro- scopes in order to clear up the terms used in catalog de- scriptions of such equipment.

Information on the history of the microscope.

Information on the history of the microscope.

History Microscope 

Although today the microscope is an accepted weapon in the armamentarium both of the scientist and the industrialist, its advent is relatively new. There seems no doubt that the properties of burning glasses were known to the ancients. The allusion to these in Aristophanes' comedy " The Frogs " would indicate this, but there seems equally to be no doubt that the use of burning glasses presumably flasks filled with water did not lead to the discovery of lenses, for Pliny the younger, in his treatise on eyesight, refers to various ocular diseases and their cures. He mentions presbyopia, but nowhere does he refer to its correction by means of lenses. There is also a mass of other evidence which has been fully explored by Dr. H. Martin, which conclusively debuts any assumptions of the existence and utilization of lenses in any form. The invention, if it may be so called, of spectacle lenses, is generally attributed to Salvino Degli Armati, while Alexander Spina, a monk of Pisa, is said to have divulged the secret of their construction and use. It is stated that on Armati's tomb was the inscription "Here lies the body of Salvino Armati. He invented spectacles; may God forgive his sins." This inscription is claimed to have existed and was seen in the year 1820.

Roger Bacon, the English Franciscan of Ilchester, who was a contemporary of Armati, was acquainted with the use of lenses, both convex and concave. There is no evidence whether he arrived independently at this knowledge, or through an acquaintance with Armati. From his Opus Majus it might be assumed that he had employed lenses as simple microscopes and, by stretch of imagination that he was acquainted with the telescope, but as no directions for the construction of such instruments are given, the question must be regarded as mere speculation. From the lack of evidence or of contemporary work, it may be assumed that at this period both microscopes and telescopes had yet to be discovered. From this time onwards spectacles of various types are figured in portraits of individuals and in paintings, and there is no doubt that they came into relatively general use. At the Renaissance with its intense mental stimulus the floodgates of enquiry were opened. It was a period of intellectual adventure and it may seem strange to those acquainted with
the literature of the time the questioning attitude of men's minds, the feeling that mankind stood on the brink of a new revelation that the lenses used for spectacles were not developed further.

Although the invention of the telescope and the microscope is generally attributed to the brothers Janssen of Middleburgh in Zeeland, it is only just to quote the following from Dr. Nicholson's essay: "The New Astronomy and English Literature Imagination". So far as England is concerned, there is evidence of the invention of the telescope there, even earlier than on the Continent. It is generally agreed by historians that Leonard Digges, from his study of a manuscript of Roger Bacon, had discovered a principle of the telescope about 1550, although until recently no suggestion has been made that his telescope was designed or used for astronomical purposes. "According to his son, Thomas Digges, ' Bi-concave and convex mirrors and circular and parabolic forms/ Digges" Not only discovered things far off, read letters, numbered pieces of money . .but also seven miles off declared what had been done at that very instant in private places."

Certainly Digges' illustrations justify no assumption that he was acquainted with or employed what would have presumably been an anticipation of the Newtonian form of telescope.

Borelius, who was Ambassador of the Low Countries, in his treatise "De Vero Telescopii Invent ore” has left on record a notarial declaration that the telescope was invented by the brothers Janssen, who were spectacle lens makers in Middleburgh and the date is approximately 1560. It is stated that one of the brothers, having polished a spectacle lens, decided to examine its surface by the aid of another lens. To his amazement, the church clock appeared, when seen through the two lenses, both enlarged and nearer. Even if not true, it is a pretty tale and pregnant with the age-old fact that mankind sees but does not observe.

Historically the period was interesting. Motley's epic, "The Rise -of the Dutch Republic”; provides the necessary background. There is evidence that the Janssens were members of what we to-day call the "Resistance Movement” As spectacle makers they visited all the Provincial and city fairs in the Low Countries, passing news of the anti-Spanish movement and circulating spurious coin so as to undermine confidence in Spanish currency.

On the discovery of the telescope, as good patriots, they showed their invention to the Archduke Maurice and he, recognizing its military value, purchased the exclusive right of the use of the instrument for a term of years and it would appear that the Janssens honoured this agreement. The invention of the telescope is, unfortunately, attributed to Lipperchey, but we believe this to be incorrect. At the time of the discovery of the instrument, Lipperchey appears to have been employed by the Janssens as a workman, and not being bound by the undertakings entered into by them, he, on leaving their employ, set up on his own account and commenced the sale of these instruments.

The original telescope consisted of two bi-convex lenses and objects were seen inverted. The first microscopes were in effect very short focus telescopes. It is understandable that with an extremely limited scientific world, the members of which were in correspondence with each other, information of the telescope would spread and that it would reach Galileo, who was already a well-known and famous mathematician. He did not invent the telescope, but he did substitute a concave for a convex eye lens, as a result of which objects were seen erect instead of inverted.

To trace the history of the microscope from this time onwards would necessitate the production of several volumes. The period was one fruitful in ideas and one in which design being in a fluid state, was rich with suggestions for the future. Design was, unfortunately, to become conventionalized in certain channels and the ideas adumbrated during this period of flux were only realized in their ultimate form within the last century.

From the early seventeenth century to the middle of the eighteenth century there is a voluminous literature, but the books are scarce.
Passing in rapid review the various developments in design, we would refer to Descartes. This simple microscope designed by Descartes was the forerunner of the Lieberkuhn, which had a great vogue until the 1880's. The principle was applied by Mr. E. M. Nelson to his reflecting magnifier and subsequently appeared again on the microscope in the form of ring illuminators, Ultrapak, etc., and is now in frequent use.

The microscope of Campani can be justly termed the precursor of the pocket microscopes, which have been so prominent during the past two decades. It was also the model upon which the Wilson screw barrel and similar instruments were developed. Of still greater interest is Hooke's compound microscope. For the first time coarse and fine adjustments are incorporated, and provision is made for inclination of the body tube, also the object can be orientated for examination at any angle. Admittedly the movements incorporated are rudimentary, but the germ of future design is present. For the first time a lamp with the equivalent of a condensing system is provided, and it will be noted that adjustment to the lamp condensing lens is incorporated. Incidentally, it will be remembered that Hooke was the inventor of Hooke's joint, known to all users of scientific instruments and employed with success up to the present time.

Hooke (Secretary of the Royal Society) was a man of amazing mental activity. He made attempts to overcome the chromatic and spherical aberrations in the lens systems of the time. Actually he introduced a field lens to the eyepiece, but while admitting that there was an improvement in performance, he objected that the field of view was reduced. Another attempt was the introduction of water between the objective and the eye lens system. Again he found that the continuous path of light had its advantages, but the method was inconvenient and the gain insufficient to justify this. He also experimented with the use of colour screens.

The Capucin Priest, Cherubin d'Orleans, introduced the first binocular microscope. This was a true binocular. Not only were two eyepieces provided, but also two objectives. In his own book, and Zahn, figures of these eyepieces appear and it will be seen that they were provided with inter-pupillary adjustments. No coarse focusing adjustment was fitted for this microscope, objects were focused by moving the stage nearer to or further from the objective surely the forerunner of the stage focusing used so extensively in the next century and employed to-day on metallurgical microscopes.

The microscopes of Van Leeuwenhoek have so often been figured and referred to that no illustration is necessary, nor in view of DobelFs publication, in which work the author has been extraordinarily successful in bringing Van Leeuwenhoek to life and making the reader feel that he is actually in mental contact with Dobell's hero is any reference to Van Leeuwenhoek required.

The next suggestive design in chronological order is that of Bonani, and nothing can be more suggestive or more in advance of its time. The complete instrument is virtually based upon the principle of the optical bench. There is an attempt at rigidity. The microscope body is fitted with coarse and fine adjustments, the coarse being by rack and pinion. The sub-stage or lamp condenser is provided with focusing motion, surely the prototype of the microscope used so successfully for metallurgical research, photomicrography, etc., up to within the last twenty years.

The microscope by Joblot, incorporates the first objective slide. Joblot mounted his objective in thin, brass, dovetailed slides, and these, some of them incorporating one, others two, objective lenses, were interchangeable and were, what would be known as, objective changers on the microscope.

The first revolving nosepiece was introduced either by Adams or by Martin. These two designers were contemporaries and it is sometimes difficult to attribute the various designs for which they are responsible in correct priority the one to the other. It is perhaps convenient at this point to interpolate a note on the first Scientific Society. This, under the name of " The Academy of the Lynx," was founded in Rome and met at the house of the Prince Cesi. Stelluti, Galileo and many others were members. The new knowledge obtained by means of the microscope and the telescope was of such enthralling interest and fitted in with the new outlook of the thinking section of mankind, resultant from the Renaissance. The Church had not fully determined her attitude. The Pope Urbino he who was interested in Galileo's work occupied the Papal throne,and both as a compliment and to placate him, the first monograph ever published was on the honey bee. This was selected for study because the bee figured in the papal coat of arms. It will be remembered that Milton made the Grand Tour. From his subsequent essay " Aeropagitica " it is known that he met Galileo (M. N.), and the whole of his poetry subsequent to that meeting is instinct with awe of a new knowledge of space. It is interesting to speculate upon the possibilities of Milton having attended meetings of the
Academy of the Lynx and subsequently meetings of the Royal Society, through which there is then a continuous link from the first Scientific Society to the present day, for the Royal Society has the longest continuous history of any scientific society in the world.

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General information on microscope objectives and eye pieces.

General information on microscope objectives and eye pieces.

Microscope Objectives and Eye-Pieces

Although considerable magnifying power may be attained by the use of two single lenses arranged in a compound form, there is no advantage in it, from the fact that the faults in the lenses are correspondingly magnified, and these are so considerable that they destroy what it is the purpose of the microscope to give a distinct image.

Classes of Microscope Objectives

Objectives may be divided into two classes, dry and immersion.

Dry Objectives – Microscopy

Dry objectives have no intervening medium except air exists between the cover and objective.

Immersion Objectives – Microscopy

In immersion or "wet" objectives a fluid is used to connect the upper surface of the cover to the front surface of the objective. The use of immersion fluid has several advantages, the first of which is that the objective may be made to give better performance, as will be explained later on ; the second is that more light will be transmitted, as there is less loss of it by refraction.

It should be understood however that no advantage will be gained by using immersion fluid with a dry objective. It does not increase its effectiveness one particle, on the contrary it detracts from its quality. When it is stated that an immersion objective has a greater capacity, it is with the understanding that it is so constructed as to give this result.

While many immersion objectives are constructed to work both as dry and immersion, such a plan cannot be said to be advantageous. Such objectives may be made to work well in one direction and be of indifferent quality in the other, or may be of medium grade both ways. There is no question that the best plan .is to have each objective selected with a view to a specific purpose and use it for this purpose only. There are two fluids in general use at the present time, water and homogeneous fluid. The latter expression means of the same kind, and refers to the fact that the fluid has about the same refractive and dispersive power as glass, so that when this fluid fills up the space between the two surfaces of glass, a ray of light passes through the three mediums as if they were one body.

The two large classes of dry and immersion objectives may again be subdivided into two classes objectives for long and short standard tube. As followed by some firms at present and what it is hoped will become a universal custom in time, each objective is marked for the tube-length for which it is corrected and with which it is assumed it will accomplish the best results.

Objectives are sometimes called powers, and in this sense are divided into three classes : low, medium and high powers.

Dr. Carpenter classifies them as follows:

Low powered objectives are: 3 inch, 2 inch, 1 1/2 inch, 1 inch, 2/3 and 3/4 of an inch.

Medium powered objectives: 4/10 of an inch, 1/2 inch, 1/4 inch, 1/5 inch ;

High powered objectives are: 1/6 inch, 1/8 inch, 1/10th of an inch, 1/12 inch, 1/16 inch, 1/20 inch, 1/25 inch.

Objectives – Microscope Optics

As the objective is the most important of the two optical parts, it follows that this must be as free from faults as possible and all that human ingenuity and skill can devise is utilized to attain this end. The advance in the perfection of the objective has been step by step and each era was at the time considered by many authorities the limit to further improvement. Each advance was signalized by a marked opposition and disbelief of its possibility. It is therefore of inestimable creditto the pioneer objective-makers, and notably among these two Americans, who by quiet but stubborn application disproved previous claims and opened the way to further improvements. A theoretical limit has been fixed on the capacity of the microscope, which according to our present knowledge can not even be reached.

While the introduction of water immersion made it possible to obtain higher optical results than with the dry objectives, as will be explained later on, the homogeneous immersion offers still greater possibilities in this direction, and the advantages are so pronounced that the former are gradually coming into disuse, although for certain kinds of work they will be preferred and used by many persons. At present homogeneous fluid is made of either thickened glycerine or cedar oil, and great care is required in keeping the front of objectives and cover glasses properly cleaned, in which respect water has the decided advantage.

It might be stated that such high power as 1/25 th and1/20 th were very rarely constructed, and the 1/16 th may be considered the maximum, while the 1/12 th is that most ordinarily used. This power will give all the optical advantages, while higher powers involve so many mechanical difficulties as to increase the cost of production very considerably, and as a rule detract from the optical qualities.

A modern objective of the highest capacity may be considered a work of art, and there are a few productions of the human hand which exact so much untiring application, ingenuity and skill.

Objective Systems

An objective is said to consist of systems which may vary in number from one to four and five ; two and three are however mainly in use. They are the individual portions consisting of one, two or three lenses, which when more than one, are cemented together and make up the objective. An achromatic sinP gle system may consist of two or three lenses, and a three or four system objective may consist of as many as seven or eight lenses. The systems are called in their A order : anterior or front, middle and posterior. When one consists of two lenses it is called a doublet, when of three lenses a triplet. A is the anterior, M the middle and P the posterior systems ; thus also A is a single system, M a double and P a triple one.

The various features which must be considered as determining the quality of an objective are : angular aperture, achromatism, resolving power, flatness of field, penetration, working distance and magnifying power. Although these attributes may be considered separately, some of them go hand in hand. The presence or extent of one necessarily involves or precludes another.